林业科学
林業科學
임업과학
Scientia Silvae Sinicae
2015年
7期
60-68
,共9页
松墨天牛%ATP合成酶%基因表达%RT-qPCR%RACE
鬆墨天牛%ATP閤成酶%基因錶達%RT-qPCR%RACE
송묵천우%ATP합성매%기인표체%RT-qPCR%RACE
Monochamus alternatus%ATP synthase%gene expression%RT-qPCR%RACE
【目的】研究松墨天牛 ATP合成酶 D亚基基因的鉴定及其表达特性，为开展昆虫 ATP 合成酶基因研究提供分子信息和参考，为深入研究 ATP合成酶基因在松墨天牛中的生理、毒理作用奠定基础。【方法】对从松墨天牛 cDNA文库中克隆的一条表达序列标签(EST)进行3'cDNA 末端快速扩增，将获得的扩增序列与 cDNA文库中的序列进行拼接，经开放阅读框( ORF)检索和 Blast同源比对鉴定基因；用 ProtParam tool软件分析基因编码的蛋白质性质，用 DNAMAN软件构建系统发育树，用实时荧光定量 PCR 分析基因在不同虫态、成虫各部位和幼虫组织中的表达特性。【结果】获得了1条长度为1133 bp的 cDNA，其5'端非编码区长89 bp，3'端非编码区长294 bp，ORF 长750 bp，编码 ATP 合成酶 D 亚基，命名为 MaATPSE ( GenBank 登录号：KM101044)。MaATPSE编码249个氨基酸残基，预测分子质量为28.21 kDa，等电点为9.40，稳定系数为31.13。MaATPSE 含有3个丝氨酸磷酸化位点、4个苏氨酸磷酸化位点、2个酪氨酸磷酸化位点和1个糖基化位点。松墨天牛与黑腹果蝇和家蚕的 MaATPSE相似性均为83%；松墨天牛与赤拟谷盗在系统发育树上聚成一支，相似性为87%，与其他非鞘翅目昆虫的遗传距离较远。MaATPSE在蛹和幼虫的相对表达量分别是成虫的3.70和2.65倍，各虫态之间表达有显著差异(P＜0.05)； MaATPSE在幼虫各组织中均有表达，在脂肪体中的表达量最高，为对照的5.94倍，其次是体壁和血淋巴，分别为对照的3.93和2.88倍；中肠和马氏管的表达量较低，分别为对照的0.24和0.28倍；除马氏管和中肠的相对表达量无显著差异外，各组织均有显著差异(P ＜0.05)。MaATPSE 在成虫头、腹、触角、足和翅的相对表达量分别为对照的2.24，3.09，3.52，3.39和4.45倍，胸部的表达量较低，为对照的0.68倍；成虫各部位之间相对表达量有显著差异( P＜0.05)。【结论】克隆到松墨天牛 ATP合成酶 D亚基基因MaATPSE，包括5'和3'非编码和完整的 ORF。MaATPSE 与赤拟谷盗 ATP 合成酶最为相似，MaATPSE 在松墨天牛幼虫、蛹和成虫中都有表达，在幼虫各组织和成虫各部位中广泛分布。
【目的】研究鬆墨天牛 ATP閤成酶 D亞基基因的鑒定及其錶達特性，為開展昆蟲 ATP 閤成酶基因研究提供分子信息和參攷，為深入研究 ATP閤成酶基因在鬆墨天牛中的生理、毒理作用奠定基礎。【方法】對從鬆墨天牛 cDNA文庫中剋隆的一條錶達序列標籤(EST)進行3'cDNA 末耑快速擴增，將穫得的擴增序列與 cDNA文庫中的序列進行拼接，經開放閱讀框( ORF)檢索和 Blast同源比對鑒定基因；用 ProtParam tool軟件分析基因編碼的蛋白質性質，用 DNAMAN軟件構建繫統髮育樹，用實時熒光定量 PCR 分析基因在不同蟲態、成蟲各部位和幼蟲組織中的錶達特性。【結果】穫得瞭1條長度為1133 bp的 cDNA，其5'耑非編碼區長89 bp，3'耑非編碼區長294 bp，ORF 長750 bp，編碼 ATP 閤成酶 D 亞基，命名為 MaATPSE ( GenBank 登錄號：KM101044)。MaATPSE編碼249箇氨基痠殘基，預測分子質量為28.21 kDa，等電點為9.40，穩定繫數為31.13。MaATPSE 含有3箇絲氨痠燐痠化位點、4箇囌氨痠燐痠化位點、2箇酪氨痠燐痠化位點和1箇糖基化位點。鬆墨天牛與黑腹果蠅和傢蠶的 MaATPSE相似性均為83%；鬆墨天牛與赤擬穀盜在繫統髮育樹上聚成一支，相似性為87%，與其他非鞘翅目昆蟲的遺傳距離較遠。MaATPSE在蛹和幼蟲的相對錶達量分彆是成蟲的3.70和2.65倍，各蟲態之間錶達有顯著差異(P＜0.05)； MaATPSE在幼蟲各組織中均有錶達，在脂肪體中的錶達量最高，為對照的5.94倍，其次是體壁和血淋巴，分彆為對照的3.93和2.88倍；中腸和馬氏管的錶達量較低，分彆為對照的0.24和0.28倍；除馬氏管和中腸的相對錶達量無顯著差異外，各組織均有顯著差異(P ＜0.05)。MaATPSE 在成蟲頭、腹、觸角、足和翅的相對錶達量分彆為對照的2.24，3.09，3.52，3.39和4.45倍，胸部的錶達量較低，為對照的0.68倍；成蟲各部位之間相對錶達量有顯著差異( P＜0.05)。【結論】剋隆到鬆墨天牛 ATP閤成酶 D亞基基因MaATPSE，包括5'和3'非編碼和完整的 ORF。MaATPSE 與赤擬穀盜 ATP 閤成酶最為相似，MaATPSE 在鬆墨天牛幼蟲、蛹和成蟲中都有錶達，在幼蟲各組織和成蟲各部位中廣汎分佈。
【목적】연구송묵천우 ATP합성매 D아기기인적감정급기표체특성，위개전곤충 ATP 합성매기인연구제공분자신식화삼고，위심입연구 ATP합성매기인재송묵천우중적생리、독리작용전정기출。【방법】대종송묵천우 cDNA문고중극륭적일조표체서렬표첨(EST)진행3'cDNA 말단쾌속확증，장획득적확증서렬여 cDNA문고중적서렬진행병접，경개방열독광( ORF)검색화 Blast동원비대감정기인；용 ProtParam tool연건분석기인편마적단백질성질，용 DNAMAN연건구건계통발육수，용실시형광정량 PCR 분석기인재불동충태、성충각부위화유충조직중적표체특성。【결과】획득료1조장도위1133 bp적 cDNA，기5'단비편마구장89 bp，3'단비편마구장294 bp，ORF 장750 bp，편마 ATP 합성매 D 아기，명명위 MaATPSE ( GenBank 등록호：KM101044)。MaATPSE편마249개안기산잔기，예측분자질량위28.21 kDa，등전점위9.40，은정계수위31.13。MaATPSE 함유3개사안산린산화위점、4개소안산린산화위점、2개락안산린산화위점화1개당기화위점。송묵천우여흑복과승화가잠적 MaATPSE상사성균위83%；송묵천우여적의곡도재계통발육수상취성일지，상사성위87%，여기타비초시목곤충적유전거리교원。MaATPSE재용화유충적상대표체량분별시성충적3.70화2.65배，각충태지간표체유현저차이(P＜0.05)； MaATPSE재유충각조직중균유표체，재지방체중적표체량최고，위대조적5.94배，기차시체벽화혈림파，분별위대조적3.93화2.88배；중장화마씨관적표체량교저，분별위대조적0.24화0.28배；제마씨관화중장적상대표체량무현저차이외，각조직균유현저차이(P ＜0.05)。MaATPSE 재성충두、복、촉각、족화시적상대표체량분별위대조적2.24，3.09，3.52，3.39화4.45배，흉부적표체량교저，위대조적0.68배；성충각부위지간상대표체량유현저차이( P＜0.05)。【결론】극륭도송묵천우 ATP합성매 D아기기인MaATPSE，포괄5'화3'비편마화완정적 ORF。MaATPSE 여적의곡도 ATP 합성매최위상사，MaATPSE 재송묵천우유충、용화성충중도유표체，재유충각조직화성충각부위중엄범분포。
[Objective]Mitochondrial ATP synthase exists in the plasma membranes of bacteria,thylakoid membranes of chloroplasts and the inner membranes of mitochondria,and is described as a splendid molecular machine,and a key enzyme for energy conservation in mitochondria. The proton motive force generated across the membrane by electron flow is used to drive the ATP synthesis from ADP and inorganic phosphate. The Japanese pine sawyer, Monochamus alternatus Hope ( Coleoptera: Cerambycidae ) , is a longicorn beetle that is notorious as a vector of the pinewood nematode,Bursaphelen chusxylophilus ( Steiner etBuhrer) Nickle,which causes pine wilt disease. This beetle is widely distributed in East Asian countries,including Japan,China and South Korea,vectoring the pine wilt disease there. The objective of the present study is to provide molecular information and references for further researches on insect ATP synthase gene,and physiological and toxicological functions of the ATP synthase gene of M. alternatus. [Method]An expressed sequence tag ( EST) from Monochamusalternatus cDNA Library was amplified by 3' rapid amplification of cDNA ends. The cloned gene was characterized by splicing with the EST,open reading frame( ORF) research and Blast homologous comparison. ProtParam tool and DNAMAN software were used to analyze the characteristics of deduced protein and construct phylogenetic tree,respectively. The expression characteristics of the cloned gene at different developmental stages and in different parts of adults and larval tissues of M. alternatus were analyzed by real time quantitative PCR. [Result]A cDNA with 1133bp length was cloned,containing a 89 bp at its 5'-UTR,a 294 bp at its 3'-UTR,and an ORF of 750 bp which codes for ATP synthase D subunit designated as MaATPSE (GenBank accession number: KM101044). The MaATPSE encodes 249 amino acid residues,and the deduced molecular weight,isoelectric point and stability coefficient are 28. 21 kDa,9. 40 and 31. 13,respectively. The MaATPSE contains 3 serine phosphorylation sites,4 threonine phosphorylation sites,2 tyrosine phosphorylation sites and 1 glycosylation sites. The MaATPSE has 87% identity with Tribolium castaneum, 83% identity both with Bombyx mori and Drosophila melanogaster,respectively. M. alternatus and T. castaneum are on the same branch in phylogenetic tree,and there is a long genetic distance between M. alternatus and non Coleoptera insects. The relative expression level of MaATPSE in pupae and larvae was 3 . 70 times and 2 . 65 times of that in adults,respectively. There was a significant difference in expression among different developmental states (P<0. 05). The MaATPSE was expressed in various tissues of larvae, the expression in fat body was highest,and 5. 94 times of that in control,and the expression in body wall and hemolymph ranked the second,and was 3. 93 times and 2. 88 times of that in control,respectively. The expression in midgut and Malpighian tubules was relatively low,and was 0. 24 times and 0. 28 times of that in control,respectively. There were significant differences in MaATPSE expression among various larval tissues,except that between Malpighian tubules and midgut ( P <0. 05 ) . The relative expression level in adult head,abdomen,antennae,feet,wings and thorax was 2. 24 ,3. 09 ,3. 52 ,3. 39 ,4. 45 and 0. 68 times of that in control,respectively. There were significant differences in MaATPSE expression among various parts in adults ( P < 0. 05 ) . [Conclusion]The Monochamus alternatus ATP synthase D subunit gene containing 5'-UTR,3'-UTR and a complete ORF was cloned in this study. The MaATPSE and ATP synthase of T. castaneum are the most similar at amino acid level. The MaATPSE is extensively expressed in the larvae,pupae and adults,in various tissues of larvae and parts of adults of M. alternatus.
